Nanonozzle device arrays: their preparation and use for macromolecular analysis

ABSTRACT

Constricted nanochannel devices suitable for use in analysis of macromolecular structure, including DNA sequencing, are disclosed. Also disclosed are methods for fabricating such devices and for analyzing macromolecules using such devices.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/889,256 filed Jun. 1, 2020, now U.S. patent Ser. No. 11/529,630,which is a continuation of U.S. application Ser. No. 15/801,081 filedNov. 1, 2017, now U.S. patent Ser. No. 10/676,352, which is acontinuation of U.S. application Ser. No. 14/712,816 filed May 14, 2015,now U.S. Pat. No. 9,845,238, which is a divisional of U.S. applicationSer. No. 12/374,141 filed Aug. 25, 2009, now U.S. Pat. No. 9,061,901,which is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2007/016408 filed Jul. 19, 2007 designating theU.S.; which claims the benefit of U.S. Provisional Application No.60/831,772, filed Jul. 19, 2006; U.S. Provisional Application No.60/908,582, filed on Mar. 28, 2007, and U.S. Provisional Application No.60/908,584, filed Mar. 28, 2007. The entireties of these listedapplications are incorporated by reference herein.

FIELD OF INVENTION

The present invention pertains to the field of nanoscale devices. Thepresent invention also pertains to the field of macromolecularsequencing, particularly the field of DNA sequencing andcharacterization.

BACKGROUND OF THE INVENTION

Various scientific and patent publications are referred to herein. Eachis incorporated by reference in its entirety.

Biomolecules such as DNA or RNA are long molecules composed ofnucleotides, the sequence of which is directly related to the genomicand post-genomic gene expression information of an organism. In mostcases, the mutation or rearrangement of the nucleotide sequences duringan individual's life span can lead to disease states such as geneticabnormalities or cell malignancy. In other cases, the small amount ofsequence differences among each individual reflects the diversity of thegenetic makeup of the population. Because of these differences ingenetic sequence, certain individuals respond differently toenvironmental stimuli and signals, including drug treatments. Forexample, some patients experience positive response to certain compoundswhile others experience no effects or even adverse side effects.

The fields of population genomics, medical genomics and pharmacogenomicsstudying genetic diversity and medical pharmacological implicationsrequire extensive sequencing coverage and large sample numbers. Thesequencing knowledge generated would be especially valuable for thehealth care and pharmaceutical industry. Cancer genomics and diagnosticsstudy genomic instability events leading to tumorigenesis. All thesefields would benefit from technologies enabling fast determination ofthe linear sequence of biopolymer molecules such as nucleic acids, orepigenetic biomarkers such as methylation patterns along thebiopolymers. There is a long felt need to use very little amount ofsample, even as little as a single cell. This would greatly advance theability to monitor the cellular state and understand the genesis andprogress of diseases such as the malignant stage of a cancer cell.

Most genome or epigenome analysis technologies remain too expensive forgeneral analysis of large genomic regions for a large population. Inorder to achieve the goal of reducing the genomic analysis cost by atleast four orders of magnitude, the so-called “$1000 genome” milestone,new technologies for molecular analysis methods are needed. See “TheQuest for the $1,000 Human Genome,” by Nicholas Wade, The New YorkTimes, Jul. 18, 2006.

One technology developed for fast sequencing involves the use of ananoscale pore through which DNA is threaded. Historically, the“nanopore” concept used a biological molecular device to produce ioniccurrent signatures when RNA and DNA strands are driven through the poreby an applied voltage. Biological systems, however, are sensitive to pH,temperature and electric fields. Further, biological molecules are notreadily integrated with the semiconductor processes required forsensitive on-chip electronics.

Many efforts have been since focused on designing and fabricatingartificial nanopores in solid state materials. These methods, however,which are capable of producing only pores in membranes are not capableof producing longer channels needed to achieve true single-moleculesequencing of long biological polymers such as DNA or RNA.

Accordingly, there is a need in the field for devices capable ofyielding sequence and other information for long biological polymerssuch as DNA or RNA.

SUMMARY OF THE INVENTION

In meeting the described challenges, in a first aspect the presentinvention provides methods for characterizing one or more features of amacromolecule, comprising linearizing a macromolecule residing at leastin part within a nanochannel, at least a portion of the nanochannelbeing capable of physically constraining at least a portion of themacromolecule so as to maintain in linear form that portion of themacromolecule, and the nanochannel comprising at least one constriction;transporting at least a portion of the macromolecule within at least aportion of the nanochannel such that at least a portion of themacromolecule passes through the constriction; monitoring at least onesignal arising in connection with the macromolecule passing through theconstriction; and correlating the at least one signal to one or morefeatures of the macromolecule.

In a second aspect, the present invention provides devices for analyzinga linearized macromolecule, comprising two or more fluid reservoirs; anda nanochannel comprising a constriction, the nanochannel placing the atleast two fluid reservoirs in fluid communication with one another.

Further provided are methods for transporting a macromolecule,comprising providing at least two fluid reservoirs; providing an atleast partially linearized macromolecule, at least a portion of themacromolecule residing in a nanochannel, the nanochannel placing the atleast two reservoirs in fluid communication with one another, thenanochannel comprising a constriction; and applying a gradient to themacromolecule, the gradient giving rise to at least a portion of thelinearized macromolecule being transported within at least a portion ofthe nanochannel.

Additionally provided are methods for fabricating a constrictednanochannel, comprising providing a nanochannel; the nanochannel havingan internal diameter in the range of from about 0.5 nm to about 1000 nm,and the nanochannel having a length of at least about 10 nm; reducingthe internal diameter of the nanochannel either at a location within thenanochannel, at a location proximate to the end of the nanochannel, orboth, so as to give rise to a constriction within or adjacent to thenanochannel, the constriction having an internal diameter in fluidiccommunication with the nanochannel, the nanochannel being capable ofmaintaining a linearized macromolecule in its linearized form, and thereduced internal diameter being capable of permitting the passage of atleast a portion of a linearized macromolecule.

Also disclosed are methods for linearizing a macromolecule, comprisingplacing a macromolecule in a nanochannel, at least a portion of thenanochannel being capable of physically constraining at least a portionof the macromolecule so as to maintain in linear form that portion ofthe macromolecule.

The general description and the following detailed description areexemplary and explanatory only and are not restrictive of the invention,as defined in the appended claims. Other aspects of the presentinvention will be apparent to those skilled in the art in view of thedetailed description of the invention as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawingsexemplary embodiments of the invention; however, the invention is notlimited to the specific methods, compositions, and devices disclosed. Inaddition, the drawings are not necessarily drawn to scale. In thedrawings:

FIG. 1 a is a schematic view of a DNA sequencer showing linearizeddouble-stranded DNA passing through a nanochannel into an outletreservoir, where a measurement device detects physical, chemical,electrical, or other changes in the outlet reservoir or within thenanochannel related to the passage of the DNA;

FIG. 1B depicts a DNA molecule flowing through a nanonozzle constrictionat the end of a nanochannel, wherein the DNA molecule is adouble-stranded DNA having a first strand comprising a nucleic acidsequence of SEQ ID NO: 1 and a complementary strand comprising a nucleicacid sequence of SEQ ID NO: 2;

FIG. 1 c depicts the data evolved from the passage of the DNA throughthe constricted nanochannel;

FIG. 2 a is a schematic view of a DNA sequencer showing linearizedsingle-stranded DNA passing through a nanochannel into an outletreservoir, where a measurement device detects any physical, chemical,electrical, or other changes in the outlet reservoir or within thenanochannel related to the passage of the DNA;

FIG. 2 b depicts single-stranded DNA molecule flowing through ananonozzle constriction at the end of a nanochannel;

FIG. 2 c depicts the data evolved as individual nucleotides of thesingle-stranded DNA pass through the constriction of the nanochannel;

FIG. 3 a is a schematic view of a DNA sequencer showing linearized,methylated double-stranded DNA passing through a nanochannel into anoutlet reservoir, where a measurement device detects any physical,chemical, electrical, or other changes in the outlet reservoir or withinthe nanochannel related to the passage of the DNA;

FIG. 3 b depicts the methylated double-stranded DNA molecule flowingthrough a nanonozzle constriction at the end of a nanochannel, whereinthe methylated double-stranded DNA molecule is a double-stranded DNAhaving a first strand comprising a nucleic acid sequence of SEQ ID NO: 3and a complementary strand comprising a nucleic acid sequence of SEQ IDNO: 4;

FIG. 3 c depicts the data evolved as individual nucleotides of themethylated double-stranded DNA pass through the constriction of thenanochannel;

FIG. 4 depicts an representative embodiment of an enclosed nanochannelin communication with a reservoir, the nanochannel having a constrictionof cross-sectional area smaller than remainder of nanochannel, andmacromolecules being linearized within the nanochannel and features ofinterest on the molecule being detected as they pass through theconstriction;

FIG. 5 depicts an enclosed nanochannel in communication with a secondnanochannel via a constriction of cross-sectional area smaller than bothnanochannels, macromolecules are linearized within the nanochannel andfeatures of interest on the molecule are detected as they pass throughthe constriction;

FIG. 6 a depicts the preparation of a constriction at the end of ananochannel by additive material deposition, and FIG. 6 b is a scanningelectron micrograph of an embodiment of such a constriction at the endof a nanochannel;

FIG. 7 a , FIG. 7 b and FIG. 7 c depict a representative fabrication ofa constriction at the end of a nanochannel by (FIG. 7 a ) deposition ofmaterial at the end of the nanochannel resulting in complete sealing ofthe channel; (FIG. 7 b ) self-terminating opening of the constrictionusing an acid etchant terminated by exposure to a neutralizing strongbase; and (FIG. 7 c ) the final nanonozzle device after the etchant andneutralizer are removed;

FIG. 8 a , FIG. 8 b and FIG. 8 c depict a representative fabrication ofa constriction at the end of a nanochannel using sacrificial material:(FIG. 8 a ) a sacrificial macromolecule is placed into the nanochanneland allowed to partially exit into the reservoir; (FIG. 8 b ) the fluidis removed and material is deposited around the sacrificial molecule;and (FIG. 8 c ) the sacrificial molecule is removed leaving aconstriction at the end of the nanochannel; and

FIG. 9 a , FIG. 9 b and FIG. 9 c present a series of three scanningelectron micrographs that describes the gradual reduction in size of achannel opening using additive deposition of material: (FIG. 9 a )additive deposition of silicon oxide on an open nanochannel of initialwidth and height of about 150 nm leads to an enclosed nanochannel ofabout 50 nm diameter, (FIG. 9 b ) variation of the deposition ofparameters leads to smaller enclosed channels, and (FIG. 9 c ) byextension, a sub-10 nm opening can be created.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Terms

As used herein, the term “substantially linear” means that theconformation of at least a portion of a long molecule, such as, but notlimited to, a polynucleic acid comprising 200 nucleic acids linkedtogether, does not loop back on itself or does not containing any sharpbends or curves greater than about 360 degrees.

As used herein, the term “nanochannel” means a conduit, channel, pipe,duct, or other similar structure having at least one nanoscaledimension.

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific devices,methods, applications, conditions or parameters described and/or shownherein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed invention. Also, as used in thespecification including the appended claims, the singular forms “a,”“an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. The term “plurality”, as usedherein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. All ranges areinclusive and combinable.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges include each and every value within that range.

In one aspect, the present invention provides methods for characterizingone or more features of a macromolecule. These methods includelinearizing a macromolecule residing at least in part within ananochannel, at least a portion of the nanochannel being capable ofphysically constraining at least a portion of the macromolecule so as tomaintain in linear form that portion of the macromolecule.

Suitable nanochannels have a diameter of less than about twice theradius of gyration of the macromolecule in its extended form. Ananochannel of such dimension is known to begin to exert entropicconfinement of the freely extended, fluctuating macromolecule coils soas to extend and elongate the coils. Suitable nanochannels can beprepared according to the methods described in Nanochannel Arrays AndTheir Preparation And Use For High-Throughput Macromolecular Analysis,U.S. patent application Ser. No. 10/484,293, filed Jan. 20, 2004, theentirety of which is incorporated by reference herein.

Suitable nanochannels include at least one constriction. Suchconstrictions function to locally reduce the effective inner diameter ofthe nanochannel. Constrictions can be sized so as to permit the passageof linearized macromolecules.

The methods also include the step of transporting at least a portion ofthe macromolecule within at least a portion of the nanochannel such thatat least a portion of the macromolecule passes through the constriction.This is shown in, for example, FIGS. 1B, 2 b, and 3 b, in which DNA isshown passing through a nanochannel constriction. Constrictions can bemade, for example by depositing material at the end of a nanochannel toseal off the nanochannel, and then etching away a portion of thedeposited material until a pore much narrower than the nanochannel isproduced. This is further illustrated in FIG. 6 a , FIG. 6 b , FIG. 7 a, FIG. 7 b , FIG. 7 c , FIG. 8 a , FIG. 8 b , FIG. 8 c , FIG. 9 a , FIG.9 b and FIG. 9 c.

Where a comparatively long macromolecule is to be analyzed, the end ofthe macromolecule is delivered into one end of the nanochannel. This isaccomplished by, for example, gradient structures, that assist suchdelivery into a nanochannel; suitable gradient structures are describedin U.S. Pat. No. 7,217,562, to Cao, et al.

The methods also include monitoring at least one signal arising inconnection with the macromolecule passing through the constriction; andcorrelating the at least one signal to one or more features of themacromolecule. This is depicted in FIGS. 1 a, 2 a, and 3 a , whichdepict a schematic of monitoring a signal arising in connection with thepassage of a macromolecule through a constriction in a nanochannel.Suitable signals include, for example, electric charge signals, opticalsignals, electromagnetic signals, magnetic signals, or any combinationthereof. Electrical signals can be monitored using, for example, any ofa variety of commercially available current meters and amplifiers. Forexample, suitable signal monitoring equipment is capable of applying aconstant voltage in the range of from about a nanovolt, or a microvolt,or a millivolt, or even a volt or more across electrodes in contact withliquid within the reservoirs and nanochannel segment. Suitablemonitoring equipment is also capable of measuring current between theelectrodes many times per second. Suitable equipment will have abandwidth of at least about 100 Hertz (“Hz”, cycles per second), orabout 1 kilohertz (“kHz”), or about 10 kHz, or about 100 kHz, or about 1megahertz (“MHz”), or even about 10 megahertz. Accordingly, current canbe made once measurements are variations on the order of the nanosecond,or the microsecond, or even on the millisecond scale. Current amplitudecan be from pico seconds . . . . Translocation speed of a sDNA can bearound 40 bases per microsecond through a typical “patch clampamplifier”. Best machine today can sample once every microseconds.Axopatch 200B, Molecular Devices (www.moleculardevices.com), having abandwidth of 100 KHz, is capable of 100,000 current measurements persecond, or equivalent to 10 microseconds per current measurement of achange in the current between the electrodes connected to the two wastereservoirs.

Macromolecules suitable for the present method include polynucleotides,polynucleosides, natural and synthetic polymers, natural and syntheticcopolymers, dendrimers, surfactants, lipids, natural and syntheticcarbohydrates, natural and synthetic polypeptides, natural and syntheticproteins, or any combination thereof. DNA is considered a particularlysuitable macromolecule that can be analyzed according to the methods asdiscussed elsewhere herein.

A macromolecule analyzed according to the methods as provided hereintypically resides within a fluid. Suitable fluids include water,buffers, cell media, and the like. Suitable fluids can also beelectrolytic, acidic, or basic.

Transporting the macromolecule is accomplished by exposing themacromolecule to a gradient, the gradient suitably applied along theflow direction of a suitable nanochannel. Suitable gradients include anelectroosmotic field, an electrophoretic field, capillary flow, amagnetic field, an electric field, a radioactive field, a mechanicalforce, an electroosmotic force, an electrophoretic force, anelectrokinetic force, a temperature gradient, a pressure gradient, asurface property gradient, a gradient of hydrophobicity, a capillaryflow, or any combination thereof. An electric field is a particularlysuitable gradient.

The gradient may be temporally constant, spatially constant, or anycombination thereof. The gradient may also vary in space and time asneeded. In some embodiments, varying the gradient enables thetransportation of the macromolecule in both forward and reversedirections. In some embodiments, varying the gradient permits the sameportion of the macromolecule to be passed through the constrictionmultiple times.

Varying the gradient also enables the user to advance the macromoleculequickly through the constriction until a region of particular intereston the macromolecule is reached, in a manner analogous tofast-forwarding a cassette tape to a desired selection. Once the regionof interest is reached, the gradient may be varied so as to pass theregion of interest through the constriction at a lower speed. Thegradient may also be reversed to effect a reverse movement of themacromolecule through the restriction. This would be analogous torewinding the cassette tape to a desired position. Accordingly, “play”,“fast forward”, “rewind”, “pause” and “stop” functions can arise bycontrolling the magnitude and polarity of the gradient between thereservoirs.

Suitable signals that can be detected include a visual signal, aninfrared signal, an ultraviolet signal, a radioactive signal, a magneticsignal, an electrical signal, an electromagnetic signal, or anycombination thereof. Electrical signals are considered preferable forthe reason that they are easily monitored, but other signals may beeffectively monitored.

The macromolecule may include one or more labels; suitable labelsinclude electron spin resonance molecule, a fluorescent molecule, achemiluminescent molecule, an isotope, a radioisotope, an enzymesubstrate, a biotin molecule, an avidin molecule, an electricalcharge-transferring molecule, a semiconductor nanocrystal, asemiconductor nanoparticle, a colloid gold nanocrystal, a ligand, amicrobead, a magnetic bead, a paramagnetic particle, a quantum dot, achromogenic substrate, an affinity molecule, a protein, a peptide, anucleic acid, a carbohydrate, an antigen, a hapten, an antibody, anantibody fragment, a lipid, a polymer, an electrically charged particle,a modified nucleotide, a chemical functional group, a chemical moiety,or any combination thereof. In some embodiments, the label is a chemicalmoiety such as a methyl group. This is shown in non-limiting fashion atFIG. 3 b , which depicts a DNA strand labeled with several methyl groupsand the detection of those methyl groups as indicative of the presenceof one or more particular features of the labeled DNA.

Signals are, in some embodiments, inherently emitted by themacromolecule. Such inherently emitted signals include magnetic signalsor radioactive signals, where the macromolecule or portions of themacromolecule are magnetic or radioactive. Where the signal isinherently emitted by the macromolecule, it may not also be necessary toilluminate the macromolecule so as to elicit a detectable signal.

In other embodiments, the signal is generated by illuminating themolecule. Illumination includes exposing at least a portion of themacromolecule to visible light, ultraviolet light, infrared light,x-rays, gamma rays, electromagnetic radiation, radio waves, radioactiveparticles, or any combination thereof. Suitable illumination devicesinclude coherent and incoherent sources of light which can illuminate,excite, or even scatter from the macromolecule. UV, VIS, IR lightsources can be used, such as lasers and other light surfaces.

Features of macromolecules detected by the disclosed methods include thesize of the macromolecule, the molecular composition of themacromolecule, the molecular sequence of the macromolecule, anelectrical property of one or more molecules of the macromolecule, achemical property of one or more molecules of the macromolecule, aradioactive property of one or more molecules of the macromolecule, amagnetic property of one or more molecules of the macromolecule, or anycombination thereof.

As discussed elsewhere herein, macromolecules are, in some embodiments,labeled. Accordingly, a feature of such macromolecule is the location ofone or more labels of the macromolecule.

The molecular composition of a molecule is also characterized by theinstant methods. The molecular composition includes the position of oneor more molecules of the macromolecule, DNA polymorphisms, DNA copynumber polymorphisms, amplifications within DNA, deletions within DNA,translocations within DNA, inversions of particular loci within DNA, thelocation of a methyl group within the macromolecule, or any combinationthereof Polymorphisms are, in some embodiments, detected by observingthe presence of a labeled probe that is complementary only to thatpolymorphism.

The detection of binding sites between a drug and the macromolecule,macromolecule-drug complexes, DNA repairing sites, DNA binding sites,DNA cleaving sites, SiRNA binding sites, anti-sense binding sites,transcription factor binding sites, regulatory factor binding sites,restriction enzyme binding sites, restriction enzyme cleaving sites, orany combination thereof are all included within the present invention.

As discussed elsewhere herein, such features are determined byinterrogating the macromolecule for the presence of one or more probescomplementary to the features of interest. The methods are shownschematically in FIGS. 1 c 2 c, and 3 c , each of which depicts themonitoring of a signal arising in connection with the passage of themacromolecule through the nanochannel constriction. In some embodiments,two or more probes are used to determine two or more features of a givenmacromolecule.

Certain embodiments of the device include a plurality of nanochannels.Such arrays of nanochannels are useful in efficiently characterizingmultiple features of a single macromolecule or multiple features ofmultiple macromolecules. It will be apparent to one having ordinaryskill in the art that labels complementary to certain features can bechosen and then applied to a given macromolecule that is thencharacterized to determine whether such features are present on thatgiven macromolecule.

Also disclosed are devices for analyzing a linearized macromolecule.Suitable devices include two or more fluid reservoirs and a nanochannelcomprising a constriction and the nanochannel placing the at least twofluid reservoirs in fluid communication with one another. As describedelsewhere herein, suitable nanochannels are capable of physicallyconstraining at least a portion of macromolecule so as to maintain thatportion in linear form. This is set forth in further detail in U.S.Application No. 60/831,772, filed Jul. 19, 2006; U.S. Application No.60/908,582, filed on Mar. 28, 2007, and U.S. Application No. 60/908,584,filed Mar. 28, 2007, the entirety of each of the aforementioned patentapplications is incorporated by reference herein. One or more of thefluidic nanochannel segments can, in some cases be curved in form,tortuous in form, or even have a varying cross-sectional dimension. Itis contemplated that not all nanochannels are equivalent incross-sectional dimension; in some case, at least one of the fluidicnanochannel segments comprises a cross-sectional dimension that isdifferent than the cross-sectional dimension of another of the fluidicnanochannel segments. Suitable devices with reservoirs can be made usingstandard silicon photolithographic and etching techniques. Nanochannellength can also be controlled using such techniques. Reservoirs andassociated microfluidic regions, including the microfluidic interfacingregions, can be sealed using a standard wafer (Si wafer-Si wafer)bonding techniques, such as thermal bonding, adhesive bonding of atransparent lid (e.g., quartz, glass, or plastic).

The constriction suitably resides at one end of the nanochannel. In someembodiments, however, the constriction resides within the nanochannel.The ultimate location of the constriction will depend on the user'sneeds. Constrictions inside a nanochannel can be made as follows: usinga sacrificial material as a filler as described further herein (see,e.g., Example 7 discussed further below).

Suitable nanochannels have a length in the range of at least about 10nm, or at least about 15 nm, or at least about 20 nm, at least about 30nm, at least about 50 nm, or even at least about 100 nm, at least about500 nm, or at least about 1000 nm. In some embodiments, the nanochannelcomprises a length at least equal to about the length of the linearizedmacromolecule.

Suitable nanochannels also have an effective inner diameter in the rangeof from about 0.5 nm to about 1000 nm, or in the range of from about 10nm to about 500 nm, or in the range of from about 100 nm to about 300nm, or in the range of from about 150 nm to about 250 nm. Nanochanneleffective inner diameters can also be at least about 15 nm, or at leastabout 20 nm, or at least about 30 nm, or at least about 40 nm, or atleast about 50 nm, or at least about 60 nm, or at least about 70 nm, orat least about 80 nm, or at least about 90 nm, or at least about 100 nm.As discussed, the nanochannel comprises an effective inner diametercapable of maintaining the macromolecule in linearized form.

The terms “effective inner diameter” and “inner diameter” are usedinterchangeably unless indicated otherwise. The term “effective innerdiameter” refers not only to nanochannels having a circularcross-sectional area, but also to nanochannels having non-circularlyshaped cross sectional areas. For example, the “effective innerdiameter” can be determined by assuming the nanochannel has a circularcross section, and forcing the actual cross sectional area of thenanochannel to be effectively calculated in terms of the area of acircle having an effective inner diameter: Cross Sectional Area ofNanochannel=pi×(effective inner diameter/2)². Accordingly, the effectiveinner diameter of a nanochannel can be determined as:

Effective Inner Diameter=2[(Cross Sectional Area ofNanochannel)/pi]^(1/2)

Constrictions suitably have an effective inner diameter or effectivedimension permitting molecular transport in the range of from about 0.5nm to about 100 nm, or in the range of from about 1 to about 80 nm, orin the range of from about 5 to about 50 nm, or in the range of fromabout 8 nm to about 30 nm, or in the range of from about 10 nm to about15 nm. Suitable constrictions have an effective inner diameter capableof maintaining a linearized macromolecule passing across theconstriction in linearized form. The effective inner diameter ordimension can be controlled by controlling the etching conditions, or bycontrolling the size of the sacrificial material within the constrictionregion.

The disclosed devices also include a gradient, such gradients suitablyexisting along at least a portion of the nanochannel. Suitable gradientsinclude an electroosmotic field, an electrophoretic field, capillaryflow, a magnetic field, an electric field, a radioactive field, amechanical force, an electroosmotic force, an electrophoretic force, anelectrokinetic force, a temperature gradient, a pressure gradient, asurface property gradient, a capillary flow, or any combination thereof.

In some embodiments, the gradient is capable of linearizing at least aportion of a macromolecule residing within at least a portion of thenanochannel. In preferred embodiments, however, the gradient is capableof transporting at least a portion of a macromolecule located within thenanochannel along at least a portion of the nanochannel.

The devices suitably include a gradient generator capable of supplyingthe described gradient. Suitable generators include a voltage source, amagnet, an acoustic source, a pressure source, or any combinationthereof.

The gradient generator is suitably capable of applying a constantgradient. The gradient generator is also, in some embodiments, capableof applying a variable gradient. The examples set forth elsewhere hereinprovide additional detail. It will be apparent to one having ordinaryskill in the art that the intensity and variability of the gradient willbe chosen according to the user's needs.

The two or more fluid reservoirs of the device comprise the same fluidin some embodiments. In other embodiments, the two or more fluidreservoirs comprise different fluids. In some embodiments, the differentfluids may be used to themselves provide the gradient used to transportthe macromolecule within the nanochannel—fluids of differing ionicstrength or other property may be chosen to provide such a gradient.Suitable fluids include buffers, acids, bases, electrolytes, cell media,surfactants, and the like.

The devices also include a detector capable of detecting a signalarising from at least a portion of the linearized macromolecule passingthrough the constriction. Suitable detectors include a charge coupleddevice (CCD) detection system, a complementary metal-oxide semiconductor(CMOS) detection system, a photodiode detection system, aphoto-multiplying tube detection system, a scintillation detectionsystem, a photon counting detection system, an electron spin resonancedetection system, a fluorescent detection system, a photon detectionsystem, an electrical detection system, a photographic film detectionsystem, a chemiluminescent detection system, an enzyme detection system,an atomic force microscopy (AFM) detection system, a scanning tunnelingmicroscopy (STM) detection system, a scanning electron microscopy (SEM)detection system, an optical detection system, a nuclear magneticresonance (NMR) detection system, a near field detection system, a totalinternal reflection (TIRF) detection system, a patch clamp detectionsystem, an electrical current detection system, an electricalamplification detection system, a resistance measurement system, acapacitive detection system, and the like.

Suitable detectors are capable of monitoring one or more locationswithin one or more of the fluid reservoirs, or, in other embodiments,are capable of monitoring a location within the nanochannel, or even alocation proximate to an end of the nanochannel. It will be apparent tothe user that employing one or more detectors capable of detectingdifferent signals at different locations would enable characterizationof multiple features of a given macromolecule.

In some embodiments, the devices include an illuminator. Illuminatorssuitable include a laser, a source of visible light, a source ofradioactive particles, a magnet, a source of ultraviolet light, a sourceof infrared light, or any combination thereof. The illuminator is used,in some embodiments, to excite a portion of the macromolecule within thenanochannel. As a non-limiting example, a source of light of a certainwavelength is used to excite fluorescent labels residing at certain,specific locations on the macromolecule so as to elicit the presence ofsuch labels.

The devices also suitably include a data processor. In some embodiments,the devices include data recorder. Such processors are used tomanipulate and correlate large data sets.

One embodiment of the disclosed devices is shown in FIG. 4 , whichdepicts a nanochannel constricted at one end proximate to a reservoirand a detector monitoring a signal arising out of a reservoir into whicha macromolecule is linearly transported. Another embodiment is shown inFIG. 5 , in which a constriction connects two nanochannels. In FIG. 5 ,it is seen that the detector monitors one or more signals evolved acrossthe constricted nanochannel assembly.

As discussed elsewhere herein, where a comparatively long macromoleculeis to be analyzed, one end of the macromolecule is first transportedinto one end of the nanochannel. As discussed, this may be accomplishedby, for example, gradient structures, that assist such delivery;suitable gradient structures are described in U.S. Pat. No. 7,217,562,to Cao, et al., the entirety of which is incorporated by referenceherein.

Also disclosed are methods for transporting a macromolecule. Suchmethods include providing at least two fluid reservoirs and providing anat least partially linearized macromolecule at least a portion of themacromolecule residing in a nanochannel. As discussed elsewhere herein,the macromolecule may be linearized by confinement in asuitably-dimensioned nanochannel having an inner diameter of less thanabout twice the radius of gyration of the linearized macromolecule.

Suitable nanochannels place the reservoirs in fluid communication withone another. Suitable nanochannels, as described elsewhere herein, alsoinclude a constriction. The dimensions of suitable constrictions aredescribed elsewhere herein.

The methods also include the application of a gradient to themacromolecule. The gradient suitably gives rise to at least a portion ofthe linearized macromolecule being transported within at least a portionof the nanochannel. Suitable gradients include an electroosmotic field,an electrophoretic field, capillary flow, a magnetic field, an electricfield, a radioactive field, a mechanical force, an electroosmotic force,an electrophoretic force, an electrokinetic force, a temperaturegradient, a pressure gradient, a surface property gradient, a capillaryflow, or any combination thereof. The gradient may suitably by constantor vary, depending on the needs of the user.

The reservoirs are generally larger in volume than that of thenanochannel segments. The reservoirs can be of almost any shape andsize. For example, a reservoir may be circular, spherical, rectangular,or any combination thereof. The size of a reservoir will be dictated bythe user's needs and may vary.

The nanochannels of the disclosed method have a length of greater thanabout 10 nm, or greater than about 12 nm, or greater than about 14 nm,or greater than about 16 nm, or greater than about 18 nm, or greaterthan about 20 nm, or greater than about 25 nm, or greater than about 30nm, or greater than about 35 nm, or greater than about 40 nm, or greaterthan about 45 nm. In some embodiments, the nanochannels have a length ofgreater than about 100 nm or even greater than about 500 nm. Suitablenanochannels can also be greater than about 1 micron in length, orgreater than about 10 microns, or greater than about 100 microns, orgreater than about 1 mm, or even greater than about 10 mm in length. Insome embodiments, the length of the nanochannel is chosen to exceedabout the length of the linearized macromolecule.

Further disclosed are methods for fabricating constricted nanochannels.These methods first include providing a nanochannel. Suitablenanochannels have an effective internal diameter in the range of fromabout 0.5 nm to about 1000 nm, or in the range of from about 1 nm toabout 500 nm, or in the range of from about 5 nm to about 100 nm, or inthe range of from about 10 nm to about 15 nm. Nanochannel effectiveinner diameters can also be at least about 15 nm, or at least about 20nm, or at least about 30 nm, or at least about 40 nm, or at least about50 nm, or at least about 60 nm, or at least about 70 nm, or at leastabout 80 nm, or at least about 90 nm, or at least about 100 nm.

The methods also include the step of reducing the effective internaldiameter of the nanochannel either at a location within the nanochannel,at a location proximate to the end of the nanochannel, or both, so as togive rise to a constriction within or adjacent to the nanochannel, theconstriction having an internal diameter in fluidic communication withthe nanochannel. Sample constrictions are shown in FIG. 1B, FIG. 4 ,FIG. 5 , FIG. 6 a , and FIG. 6 b . Suitable nanochannels are capable ofmaintaining a linearized macromolecule in its linearized form; asdiscussed elsewhere herein, this is suitably accomplished by usingnanochannel segments of suitable dimensions so as to physicallyconstrain the macromolecule to maintain the macromolecule insubstantially linear form. The reduced internal diameter of theconstricted nanochannel is suitably capable of permitting the passage ofat least a portion of a linearized macromolecule.

In one embodiment, the internal diameter of the nanochannel is reducedso as to form the constriction by additively depositing one or morematerials within, or exterior to, the nanochannel. This is suitablyaccomplished by sputtering, spraying, physical vapor deposition,chemical vapor deposition, or any combination thereof. In someembodiments, the additive deposition ceases before the nanochannel iscompletely occluded. This is depicted in FIGS. 6 a and 6 b , where thedeposition of additive material proximate to one end of a nanochannel isshown, the deposition ceasing before the nanochannel is completelyoccluded. Such a nanochannel is also shown in FIG. 9 a , FIG. 9 b andFIG. 9 c , in which the reduction in effective inner diameter is seen asthe end of the nanochannel (FIG. 9 a ) is reduced by varied deposition(FIGS. 9 b and 9 c ) of additive material.

In other embodiments of the invention, the additive deposition ceasesafter the nanochannel is completely occluded. In these embodiments, themethods include the step of reopening the sealed nanochannel by removingat least a portion of the deposited additive material. This is suitablyaccomplished by etching. The etching process entails contacting one sideof deposited additive material with an first species capable of etchingthe deposited additive material and contacting the other side of thedeposited additive material with a second species capable of retardingthe etching activity of the etch species upon contact with the firstspecies. In some embodiments, the second species is capable of haltingthe etching activity of the etch species upon contact with the firstspecies. This is depicted in FIG. 7 a , where sealing material isapplied at one end of a nanochannel and then, FIG. 7 b , etched away bya strongly basic solution, the etching ceasing, FIG. 7 c , when thebasic solution etches through the sealing material and is neutralized bythe strong acid residing on the opposite side of the sealing material.As will be apparent to those having ordinary skill in the art, theconditions in the nanochannel, the relative amounts of the first andsecond species, and other operating parameters will be adjusted so as toachieve the desired diameter.

In still other embodiments, reducing the internal diameter of thenanochannel includes several steps: (FIG. 8 a ) placing a sacrificialmaterial within the nanochannel, (FIG. 8 b ) depositing additivematerial proximate to the sacrificial material so as to fully occludethe nanochannel, and (FIG. 8 c ) selectively removing at least a portionof the sacrificial material while leaving essentially intact theproximate additive material so as to give rise to a reduced internaldiameter of the nanochannel of the dimension of the removed sacrificialmaterial. It will be apparent to those having ordinary skill in the artthat this aspect of the present invention is useful for fabricatingvoids having a variety of dimensions within nanoscale and largerchannels or within other structures. DNA and carbon nanotubes are bothconsidered suitable sacrificial materials. Other materials that may beselectively dissolved or etched away will be apparent to those havingordinary skill in the art.

The present invention also provides methods for linearizingmacromolecules so as to constrain the degrees of freedom of themacromolecules from three dimensions to essentially one dimension. Thesemethods include placing a macromolecule in a nanochannel, at least aportion of the nanochannel being capable of physically constraining atleast a portion of the macromolecule so as to maintain in linear formthat portion of the macromolecule.

The nanochannels suitably include a constriction. Suitable dimensionsfor constrictions are described elsewhere herein.

The methods also include, in some embodiments, applying a gradient tothe macromolecule, such that at least a portion of the macromoleculepasses, linearly, through the nanochannel constriction. Suitablegradients include an electroosmotic field, an electrophoretic field,capillary flow, a magnetic field, an electric field, a radioactivefield, a mechanical force, an electroosmotic force, an electrophoreticforce, an electrokinetic force, a temperature gradient, a pressuregradient, a surface property gradient, a capillary flow, or anycombination thereof.

The microchannels of the disclosed methods suitable place two or morefluid reservoirs in fluid communication with one another.

The nanochannels suitable include an internal diameter of less thanabout two times the radius of gyration of the linear conformation of themacromolecule. Nanochannels suitably have lengths of at least about 10nm, of at least about 50 nm, of at least about 100 nm, of at least about500 nm. Suitable inner diameters for nanochannels are in the range offrom about 0.5 nm to about 1000 nm, or in the range of from about 5 nmto about 200 nm, or in the range of from about 50 nm to about 100 nm.

EXAMPLES AND OTHER ILLUSTRATIVE EMBODIMENTS

The following are non-limiting examples and illustrative embodiments,and do not necessarily restrict the scope of the invention.

General Procedures. Deposition of filling material was provided bysputtering, CVD, e-beam evaporation with a tilted sample wafer atvarious angles. This step was used to both reduce the nanochannelopenings and create a tapered nozzle at the end of the channels.

Typically, to fabricate enclosed nanochannels, 100-340 nm of SiO₂ wasdeposited onto the channel openings. Effective sealing was achieved withvarious deposition conditions that were tested. At gas pressure of 30mTorr, RF power of −900 W, and DC bias of 1400 V, a deposition rate of˜9 nm/min was achieved. At lower pressure of 5 mTorr, the depositionrate was increased to an estimated 17 nm/min. Filling material wasdeposited on the nanochannel opening by sputtering at 5 mTorr. Furtherdetails about making nanochannel arrays and devices can be found in U.S.Patent Application Pub. Nos. US 2004-0033515 A1 and US 2004-0197843 A1,the entirety of which is incorporated by reference herein.

Example 1: A silicon substrate was provided having a plurality ofparallel linear channels having a 150 nm trench width and a 150 nmtrench height. These channel openings are sputtered at a gas pressure of5 mTorr according to the general procedures given above. The sputterdeposition time was 10-25 minutes to provide a nanochannel array thatcan either be partially sealed or completely sealed.

Example 2: This example provides an enclosed nanochannel array using ane-beam deposition technique. A substrate can be provided as inExample 1. Silicon dioxide can be deposited by an e-beam (thermo)evaporator (Temescal BID-1800) onto the substrate. The substrate can beplaced at various angles incident to the depositing beam from thesilicon dioxide source target; the deposition rate can be set to about 3nm/minute and 150 nm of sealing material can be deposited in about 50minutes. The angle of the incident depositing beam of sealing materialcan be varied to reduce the channel width and height to less than 150 nmand 150 nm, respectively, and to substantially seal the channels byproviding shallow tangential deposition angles.

Example 3: In this example, a nanochannel array can be contacted with asurface-modifying agent. A nanochannel array made according to Example 1can be submerged in solution to facilitate wetting and reducenon-specific binding. The solution can contain polyethelyene glycolsilane in toluene at concentrations ranging from 0.1-100 mM and remainsin contact with the nanochannel array from about 1 hour to about 24hours. Subsequent washing in ethanol and water is used to removeungrafted material.

Example 4: This example describes a sample reservoir with a nanochannelarray for a nanofluidic chip. A nanochannel array having 100 nm wide,100 nm deep nanochannels was made according to general procedures ofExample 1. The nanochannel array was spin-coated with a photoresist suchas AZ-5214E and patterned by photolithography with a photomask using aKarl Suss MA6 Aligner to provide regions on opposite ends of the channelarray for preparing the reservoirs. The exposed areas were etched usingreactive ion etching in a Plasma-Therm 720SLR using a combination of CF₄and O₂ at a pressure of 5 mTorr and RF power of 100 W with an etch rateof 20 nm/min to expose the nanochannel ends and to provide a micron-deepreservoir about a millimeter wide on the opposite ends of the channelsat the edge of the substrate.

Example 5: This example describes filling a nanofluidic chip with afluid containing DNA macromolecules to analyze DNA. A cylindrical-shapedplastic sample-delivery tube of 2 mm diameter was placed in fluidcommunication with one of the reservoirs of the nanochannel array ofExample 3. The delivery tube can be connected to an external sampledelivery/collection device, which can be in turn connected to apressure/vacuum generating apparatus. The nanochannels are wetted usingcapillary action with a buffer solution. A buffer solution containingstained for example lambda phage macromolecules (lambda DNA) wereintroduced into the nanochannel array by electric field (at 1-50 V/cm);the solution concentration was 0.05-5 microgram/mL and the lambda DNAwas stained at a ratio of 10:1 base pair/dye with TOTO-1 dye (MolecularProbes, Eugene, Oreg.). This solution of stained DNA was diluted to0.01-0.05 microgram/mL into 0.5×TBE (tris-boroacetate buffer at pH 7.0)containing 0.1M of an anti-oxidant and 0.1% of a linear polyacrylamideused as an anti-sticking agent.

Example 6: This example describes the fabrication of a nanozzle at endof nanochannel using acid etching. Fabrication of the nanozzle devicebegins with a completed silicon nanochannel having enclosed nanochannelsas provided in Example 1. Creation of the nanopore proceeds by sputtercoating a thin layer of chromium over the exposed end of the channel.Sputter coating using a Temescal system can be controlled with sub-nmprecision with deposition amounts of 5-200 nm at a rate of 0.01-1 nm/secor until the end of the nanochannel is completely covered with chromium.A wet-etch process can then be employed to open a sub-10 nm pore in thechromium. A dilute chromium etchant such as Cr⁻⁷ can be flowed into thechannel using capillary forces or other forms of pumping. Dilution canrange from 1× to 10,000×. Because Cr⁻⁷ is a highly selective acidetchant, it will preferentially react with the chromium at the end ofthe channel rather than the silicon channels themselves. To stop theetch once a pore has opened, the area outside the channel will be filledwith a highly concentrated base solution (such as sodium hydroxide) thatwill rapidly neutralize the weak acid upon breakthrough. Aftersubsequent washing of the device, the result will be a nanoscale nozzleat the end of a nanochannel.

Example 7: This example describes how to fabricate a nanonozzle chipusing a sacrificial macromolecule. A nanofluidic chip with input/outputfluid reservoirs connected by nanochannels is used to linearize doublestrand DNA (FIG. 8 a ). In this example, long fragments of DNA,approximately 1-10 Mbp in length, uniformly stained with YOYO-I ispreferable. The concentration of the DNA should be around 0.5micrograms/mL, with the dye stained to a ratio of 10:1 base pair/dye.The buffer solution is composed of 0.5×TBE (tris-boroacetate buffer atpH 7.0) containing 0.1M of an anti-oxidant and 0.1% of a linearpolyacrylamide used as an anti-sticking agent. Using the nanofluidicchip and procedure described previously (Cao 2002), the solution ofsacrificial DNA molecules are flowed into the nanochannels of the chip,where they exit the nanochannels at the outlet reservoir. Using afluorescent imaging microscope, the exit of the DNA molecules isobserved in real time, and their movement controlled by applying anelectric field across the reservoirs (1-50 V/cm). With such a scheme, adesired DNA fragment's position can be suspended having only partiallyexited the nanochannel. The nanochannel chip is then dried at 50° C. invacuum environment removing any residual buffer solution, so that theDNA fragment of interest remains partially inside the nanochannel.Interaction between the nanochannel surface and the DNA fragment, suchas through van der Waals bonding, maintains the fragment's position inthe channel during the drying process.

After the nanochannel chip has been dried, a material such as silicondioxide is deposited over the surface of the chip (FIG. 8 b ) such thatthe entrance to the nanochannel becomes blocked, and the DNA fragmentenclosed. The rate of material deposition and the temperature of thedeposition must be carefully chosen such that the DNA fragment is notdamaged during this process. Evaporating material at 0.2 A/s or less ona sample kept at −1600 C using a cooling stage has been shown to protectsmall organic molecules from damage (Austin 2003). In order to obtainuniform coverage around the nanochannel to properly form a nanonozzle,the stage is rotated and tilted during the evaporation process. Tocompletely close a nanochannel of 80 nm in diameter, approximately 200nm of silicon dioxide material should be evaporated.

Example 8: Operation of a Device—Electrical measurement for Sequencingsingle-stranded nucleic acid: A voltage bias is applied across ananochannel device having a constriction (either nanogate or nanozzle)approximately 1.5 nm inner diameter placed at one end of thenanochannel, the nanochannel being about 200 microns in length, for asingle strand nucleic acid for sequencing using electrodes (can becopper, silver, titanium, platinum, gold, or aluminum) contactingreservoirs at each end of the nanochannel, the reservoirs havingdimensions of about 5-100 microns in diameter and 1-2 microns deep. Theelectrodes are deposited into the reservoirs and lead lines leading tooutside the fluidic region for connection to a current monitor. Avoltage range of 100 mV-100V can be used. Biological buffer (TE, TBE,TAE) is placed in each reservoir, capillary action and a pressuredifferential aids in wetting the nanofluidic device using a suitablefluidic delivery pump or syringe. A nucleic acid sample (e.g., 100 basesDNA and up, at least 1000 bases, or 10000 bases, or 100000 bases, or 1million, or even 10 million, or even 100 million, up to chromosomallength) in buffer solution (1 nanoliter up to about 100 microliters) isdelivered to one or both of the reservoirs. A gradient is applied to aidin the transport of one or more polynucleic acid molecules into thenanochannel in into the constriction. A field is applied, specificallyin this example, a controlled voltage gradient to apply a current fromone reservoir, through the nanochannel, through the constriction withthe polynucleic acid residing within the constriction, and into thesecond reservoir. The electrical current flowing through this system isdetected and amplified using an Axopatch 200B (Molecular Devices) patchclamp amplifier. Typical measured currents range from about 100 fA toabout 1 uA with fluctuations approximately hundreds of picoamps as theDNA moves through the constriction. Labels attached to the single strandDNA can produce additional current fluctuations of magnitude smallerthan that created by the DNA itself. For the case of measurements with aspatial resolution of a single base, typical translocation speed is suchthat the measurement system can register a minimum of 1 measurement perbase. In the case of the Axopatch 200B with 100 kHz bandwidth, themaximum translocation speed is 100 kB/sec, assuming 50% stretching ofthe DNA molecule in the nanochannel. This gives rise to a translocationspeed of the DNA through the construction to be about 0.015 nm/microsec.The measure current differences are measured and correlated to a set ofcalibration standards to give rise to the sequence of the DNA sample.

A sample table tabulating suitable cross-sectional dimensions for theanalysis of various target molecules is shown below:

TABLE Minimum cross- sectional dimension Target Molecule Analyzed ofconstriction (nm) ss-DNA 1.5 ss-DNA + complementary strands 2 ds-DNAwith nick, gap or lesion 2 ds-DNA 2 ds-DNA + moiety (e.g. Methyl group,labeling 2.1 group) ds-DNA + small compound 2.5 ds-DNA + 3rd strandprobe 3.5 ds-DNA + biotin 5 ds-DNA + protein bound factors (e.g.  4-15Transcription factors) ds-DNA + bead (e.g. Quantum dot, magnetic 10-50beads)

1.-85. (canceled)
 86. A method for characterizing one or more featuresof a macromolecule, comprising: linearizing a macromolecule residing atleast in part within a nanochannel, the nanochannel comprisingnanochannel segments capable of physically constraining at least aportion of the macromolecule so as to maintain in linear form thatportion of the macromolecule, wherein at least one of the nanochannelsegments comprises a cross-sectional dimension that is different thanthe cross-sectional dimension of another of the fluidic nanochannelsegments, and wherein the nanochannel segments: are curved in form; aretortuous in form; and/or have a varying cross-sectional dimension; andthe nanochannel segments comprising at least one constriction;transporting at least a portion of the macromolecule within at least aportion of the nanochannel segments such that at least a portion of themacromolecule passes through the constriction; monitoring at least onesignal arising in connection with the macromolecule passing through theconstriction; and correlating the at least one signal to one or morefeatures of the macromolecule.
 87. The method of claim 86, wherein themacromolecule comprises a polynucleotide, a polynucleoside, a polymer, acopolymer, a dendrimer, a surfactant, a lipid, a carbohydrate, apolypeptide, a protein, or any combination thereof.
 88. The method ofclaim 86, wherein a feature of the macromolecule comprises the size ofthe macromolecule, the molecular composition of the macromolecule, themolecular sequence of the macromolecule, an electrical property of oneor more molecules of the macromolecule, a chemical property of one ormore molecules of the macromolecule, a radioactive property of one ormore molecules of the macromolecule, a magnetic property of one or moremolecules of the macromolecule, or any combination thereof.
 89. Themethod of claim 88, wherein the molecular composition of themacromolecule comprises the position of one or more molecules of themacromolecule, DNA polymorphisms, DNA copy number polymorphisms,amplifications within DNA, deletions within DNA, translocations withinDNA, inversions of particular loci within DNA, the location of a methylgroup within the macromolecule, or any combination thereof.
 90. Themethod of claim 86, wherein the macromolecule resides within a fluid.91. The method of claim 86, wherein the transporting comprises exposingthe macromolecule to a gradient, wherein the gradient comprises anelectroosmotic field, an electrophoretic field, a magnetic field, anelectric field, an electromagnetic field, a flow field, a radioactivefield, a mechanical force, an electroosmotic force, an electrophoreticforce, an electrokinetic force, a temperature gradient, a pressuregradient, a surface property gradient, a capillary flow, or anycombination thereof.
 92. The method of claim 86, wherein the signalcomprises a visual signal, an infrared signal, an ultraviolet signal, aradioactive signal, a magnetic signal, an electrical signal, anelectromagnetic signal, or any combination thereof.
 93. The method ofclaim 86, wherein the signal is inherently emitted by the macromolecule.94. The method of claim 86, wherein the signal is generated byilluminating the macromolecule.
 95. The method of claim 94, wherein theilluminating comprises exposing at least a portion of the macromoleculeto visible light, ultraviolet light, infrared light, x-rays, gamma rays,electromagnetic radiation, radio waves, radioactive particles, or anycombination thereof.
 96. The method of claim 86, wherein themacromolecule comprises one or more labels.
 97. The method of claim 96,wherein a feature of the macromolecule comprises the location of one ormore labels of the macromolecule.
 98. The method of claim 96, whereinthe one or more labels comprise an electron spin resonance molecule, afluorescent molecule, a chemiluminescent molecule, a radioisotope, andenzyme substrate, a biotin molecule, an avidin molecule, an electricalcharged transferring molecule, a semiconductor nanocrystal, asemiconductor nanoparticle, a colloid gold nanocrystal, a ligand, amicrobead, a magnetic bead, a paramagnetic particle, a quantum dot, achromogenic substrate, an affinity molecule, a protein, a peptide, anucleic acid, a carbohydrate, an antigen, a hapten, an antibody, anantibody fragment, a lipid, a polymer, an electrically charged particle,modified nucleotide, or any combination thereof.
 99. A device foranalyzing a polynucleotide macromolecule, the device comprising: two ormore fluid reservoirs joined by a fluid pathway; and a nanochannelcomprising nanochannel segments capable of physically constraining atleast a portion of the polynucleotide macromolecule so as to maintain inlinear form that portion of the polynucleotide macromolecule, wherein atleast one of the nanochannel segments comprises a cross-sectionaldimension that is different than the cross-sectional dimension ofanother of the nanochannel segments, and wherein the nanochannelsegments: are curved in form; are tortuous in form; and/or have avarying cross-sectional dimension; wherein the nanochannel segmentscomprises at least one constriction comprising an effective innerdiameter in the range of from about 0.5 nm to about 100 nm; and whereinthe nanochannel places the at least two fluid reservoirs in fluidcommunication with one another.
 100. The device of claim 99, wherein thenanochannel comprises a length of at least about 50 nm.
 101. The deviceof claim 99, further comprising a sensor associated with the devicelocated to detect a signal from at least a portion of the polynucleotidemacromolecule as it passes through the constriction.
 102. The device ofclaim 101, wherein the sensor is selected from the group consisting of acharge coupled device (CCD) detection system, a complementarymetal-oxide semiconductor (CMOS) detection system, a photo diodedetection system, a photo-multiplying tube detection system, ascintillation detection system, a photon counting detection system, anelectron spin resonance detection system, a fluorescent detectionsystem, a photon detection system, an electrical detection system, aphotographic film detection system, a chemiluminescent detection system,an enzyme detection system, an atomic force microscopy (AFM) detectionsystem, a scanning tunneling microscopy (STM) detection system, ascanning electron microscopy (SEM) detection system, an opticaldetection system, a nuclear magnetic resonance (NMR) detection system, anear field detection system, a total internal reflection (TIRF)detection system, a patch clamp detection system, an electrical currentdetection system, an electrical amplification detection system, aresistance measurement system, a capacitive detection system, and anycombination thereof.
 103. The device of claim 99, further comprising anilluminator selected from the group consisting of a laser, a source ofvisible light, a magnet, a source of ultraviolet light, a source ofinfrared light, and any combination thereof.
 104. The device of claim99, wherein the constriction is capable of maintaining a linearizedpolynucleotide macromolecule passing across the constriction in alinearized form.
 105. The device of claim 99, wherein the device furthercomprises a gradient, wherein the gradient comprises an electroosmoticfield, an electrophoretic field, a magnetic field, an electric field, anelectromagnetic field, a flow field, a radioactive field, a mechanicalforce, an electroosmotic force, an electrophoretic force, anelectrokinetic force, a temperature gradient, a pressure gradient, asurface property gradient, a capillary flow, or any combination thereof.